1. Field of the Invention
The present invention relates to the measurement of fluorescence by a fluorescence microplate reader. In particular, the present invention relates to a fluorescence validation microplate which is used to validate the reliability of the information received from a fluorescent assay microplate during its reading in a fluorescence microplate reader.
2. Description of Related Art
The growth of biological research and new pharmaceutical compound screening and in some cases, medical diagnostics has created a need for handling large numbers of test samples at one time in order to control costs and efficiently handle these large numbers of samples. A number of analytical methods are now available for high throughput screening of these samples. One of the fastest growing screening methodologies in life science is fluorescence spectroscopy. With this method, the fluorescent emission of test samples tagged with a fluorescent material or with intrinsic fluorescence are measured. Fluorescent emission measurement techniques rival, at this time, those of radioactive assay without the inherent danger of radioactive probes. Typically, large numbers of samples are processed for fluorescence emission reading by placement in a multi-well sample plate called a microplate. These microplates are typically a rectangular array of wells, usually 24 or 96 wells in typical examples, but even 384 well and 1536 well microplates are used. Typically, each well in a 96 well microplate is approximately 0.3–0.4 ml. Plates with larger numbers of wells have even smaller volumes. These microplate wells are filled with test samples and then placed in a special detector system called a fluorescence microplate reader (also called a fluorometer) for measuring the relative fluorescence emissions (normally as RFU's or relative fluorescence units).
Although fluorescence microplates and fluorometers are of great utility in automated screening, there are a number of issues connected with their use that affect the reliability of their use. A fluorometer has a series of optical devices wherein each device is positioned to correspond to a well in a microplate holding test samples. In the alternative, the fluorometer is fitted with a single optical device and either the plate, the reading device, or both is moved to the appropriate reading position. Use of the microplate on the fluorometer must involve alignment of each sample well with the optical device on the fluorometer. Alignment is typically done by physically moving either the microplate, the fluorometer optical device(s) or both. Movement of microplates or optical devices is done using stepper motors wherein the movement is guided on a certain number of (factory calibrated) steps from a “home” position. Alignment is adversely affected when one or more of the aligned components involved is shifted in position or becomes damaged. If, for any reason, the alignment is incorrect, the wells will not be centered properly, resulting in incorrect fluorescence measurements.
The optics used to measure fluorescence must avoid detecting transmission of fluorescence from one sample well as fluorescence from an adjacent well. This adjacent well detection problem is called “cross-talk”. Cross-talk is extremely undesirable because it means the photon emissions detected, by a particular optical device at a given location originated from the sample in a different well. In a worse case scenario, a particular well optical device is detecting fluorescent cross-talk from all the surrounding or adjacent wells. This can be as many as eight wells in a standard microplate set-up. In a minimum problem scenario, cross-talk only occurs from an adjacent (single) well but is still obviously undesirable.
Linearity of the sample readings is also extremely important to control. Linearity is a measure of the relationship between different amounts of fluorescence emissions in a series of wells as measured by their RFU's. A linear dose response relationship should exist between the measured RFU's and the strength of the fluorescence emissions. Linearity is an indication of the relative concentration of fluorescence emissions in the series of wells. As the optical, electronic and other components of the fluorometer age, the detection efficiency can be diminished at different RFU's and the linearity is affected especially at high and low fluorescence intensities. To a certain extent, these problems have been addressed by software programs and by advancing technologies with the readers themselves. However, these solutions do not solve all the linearity problems with fluorometers.
Another problem with the fluorometers is the necessity to select discrete wavelengths of both the excitation and emission light to measure fluorescence. This is done using interference filters, diffraction gratings, acusto-optical tunable filters or a variety of other devices. These devices are notoriously fragile, subject to damage by water, scratches, oxidation and the like causing fluorometers to give inaccurate results.
It would be useful therefore if there were a device capable of providing necessary information, input or feedback to detect, help correct or eliminate these problems for fluorometers. Specifically, what is needed and has not previously been provided by the art is a method or device for testing the validation of results obtained with a fluorometer.